Anti araf

HEK293T cells were seeded at 3.10⁵ cells per well in 6-wells slides (Millicell, Millipore) precoated with poly-L-lysine (Sigma) and transfected with pcdna3-myc-MITF and pcdna3-RAF plasmids (HA-ARAF, HA-BRAF or HA-CRAF) or empty vector as previously described. After 48 hours, cells were fixed in 4% paraformaldehyde, blocked (0.1% Triton X-100, 10% goat serum in PBS) and stained overnight at 4 °C with anti-myc antibody (Santa Cruz) and anti-ARAF (Cell Signaling) or anti-MITF (Sigma) and anti-BRAF (Santa Cruz) or anti-CRAF (BD Biosciences). Anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 or anti-rabbit Alexa Fluor 594 and anti-mouse Alexa Fluor 488 were used for detection. Fluoroshield with DAPI (Sigma) was used as mounting medium. Images were captured using a 3D/optigrid Leica fluorescent microscope. For quantification by using Image J software, the nuclear and cytosolic compartments were selected by applying an automatic threshold (Li Dark method) to the DAPI and FITC images. The nucleus-cytoplasm ratio was then computed by dividing the mean intensity of TexasRed2 (MITF) fluorescence extracted from nucleus region by the mean intensity from cytosolic regions obtained by subtracting DAPI from the FITC surface. The background intensity was measured on each TexasRed2 image and subtracted from the mean intensities before calculating the ratio.

For SDS–PAGE analysis, the membranes were blocked with 5 % milk in PBS Tween 20 (10 %) for 30 min at room temperature. Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-MITF (HPA003259, Sigma), anti-HA (3F10, Roche), anti-myc (9E10, Santa Cruz), anti-flag (M2, Sigma), anti-ARAF (4432, Cell Signaling), anti-BRAF (sc5284, Santa Cruz), anti-CRAF (610151, BD Biosciences), anti-ERK (sc93, Santa Cruz), anti-pERK (M8159, Sigma), anti-laminA/C (10298-1-AP, Proteintech), anti-MEK1 (sc219, Santa Cruz) and anti-β-actin (A1978, Sigma) antibodies. Antigen-antibody complexes were detected by horseradish peroxidase-coupled secondary antibodies followed by enhanced chemiluminescence. Signals were acquired using a cooled-CDD camera (Fusion FX Spectra, Vilber).